Taq error problems

In article <aquilla.1140757507B at sadye.emba.uvm.edu>,
aquilla at salus.med.uvm.edu (Tracy Aquilla) wrote:
> In Article <D2Jspv.H2u at info.swan.ac.uk>, G Jenkins wrote:
> >I'm using a PCR technique to detect mutations in genomic DNA
> >in order to quantitate the mutations I need to detect a very
> >small number of mutations above a Taq error background. I
> >therefore need to reduce the Taq error rate as much as possible. I
> >know about the addition of a proofreading polymerase, and am trying
> >that at present. Can anyone help with any other suggestions, e.g.
> >cycle number (I'm using 24), DNA concentration ( I'm using 10 microg's),
> >primer concentration (20 pM)?
> >Any help would be greatly appreciated
> >Gareth Jenkins
>> 1. keep MgCl2 conc. as low as possible while still giving decent yield
> 2. keep number of cycles as low as possible too
> 3. keep dNTP conc. as low as possible, and no higher than MgCl2 conc.
A speaker at a a PE/Cetus seminar also recommended :
4. keep cycles as SHORT as possible and if possible do the annealing and
extension simulataneously (to prevent "pausing" of taq at regions of
secondary structure, thus reducing misincorporation)
5. raise annealing temp as much as possible (I don't remember why, maybe it
reduces 3' mismatch?)
--
Opinions seen here are channeled to me by a Ring-Tailed Lemur named Oxnyx